Method of detecting microorganisms utilizing transparent sample container

ABSTRACT

The present invention relates to a transparent sample container containing, preferably, a liquid bacterial growth media for detecting microbacteria and a process for detecting microbacteria using this sample container. The container is optically transparent, heat resistant, and stable during storage. The container and process provide a bacterial growth medium substantially free of contamination upon prolonged storage of preferably about one year at 40° C.

FIELD OF THE INVENTION

The present invention is directed to a sample container and preferably,to a sample container containing a liquid microorganism growth media,wherein the sample container has good long term stability. Moreparticularly, the present invention is directed to a microbial detectingcontainer that is optically transparent, heat resistant, and resistantto breakage.

BACKGROUND OF THE INVENTION

A vast number of microorganisms are known, many of which are harmful tohumans. The presence of these microorganisms creates a continuing needfor reliable detection systems and methods of safely and efficientlyhandling microorganism samples.

In recent years there has been an increased incidence of mycobacterialdiseases, and particularly, tuberculosis. To address this increase ofsuch diseases in the population, numerous methods have been introducedfor improving the detection of the presence of various mycobacteria,such as, tuberculosis. A number of methods are directed to reducing thetime required for accurate detection of the microorganism.

One type of detection system relies on the visual detection of thepresence or absence of the growth of microorganisms. Antimicrobicsusceptibility tests use this kind of visual detection as an indicationof the efficacy of an antimicrobic compound. A disadvantage of this typeof test is the time requirements which can require an 18 to 24 dayincubation period before sufficient microorganism growth can bedetected. An example of this type of method is the Bauer-Kirby DiscMethod.

Another method of testing antimicrobic susceptibility uses a plasticpanel having several low volume cupulas. Each cupula contains adifferent test compound or different concentration of a test compounddried on the cupula surface. A test sample containing the suspectedmicroorganism is suspended in a testing medium and an aliquot isdelivered to the individual cupulas of the test panel. The dried reagentdissolves and the resulting solution is incubated for sufficient timefor the organisms to interact with the reagent and grow. The samples arevisually examined for the presence or absence of growth. This methodalso has the disadvantage of long incubation times thereby preventing arapid detection.

Light scattering methods have also been developed for determining thesusceptibility of microorganisms to antimicrobic compounds. Thesemethods require the use of a light scattering and detection apparatusthat are able to detect and measure the changes in size of themicroorganism colonies or changes in the number of the microorganismcolonies. Information on the antimicrobic susceptibility of variousmicroorganisms have been reported in as little as six hours. However,some microorganisms and antimicrobic compounds can require as long as 18hours to obtain reliable results.

Another method of determining antimicrobic susceptibility is based onquantifying ATP in the microorganism during incubation by abioluminescent method. Although this method can produce results in lesstime than other methods, the method can be difficult to carry outproperly to obtain accurate results.

The growth of microorganisms has also been detected by measuring changesin dissolved oxygen. Measuring dissolved oxygen often uses a suitableelectrode. However, some of the electrode systems consume oxygen therebyincreasing the difficulty of obtaining accurate measurements. Someelectrodes require an oxygen permeable membrane to prevent the electrodefrom interacting with the growth media and the microorganisms. Thissystem still consumes some oxygen and requires time for the solution toequilibrate with the electrodes.

Various methods for optical detection of changes in oxygen concentrationhave been developed that do not require the use of electrodes in thetest solution. These devices can be based on colorimetric or fluorimeticanalysis based on changes in the oxygen content of the solutions. Theseoptical processes can be performed economically and in a reasonable timeperiod. One example of a process for detecting microorganisms bydetecting changes in oxygen levels is disclosed in U.S. Pat. No.5,567,598 to Stitt et al. The process disclosed in this patent detectsand evaluates the metabolic activity of microorganisms using afluorescent compound.

Many of these methods require the use of a container or sample tube tocontain the sample during the analysis. Glass is a common material forthe sample tubes since glass is typically non-reactive with the samplesor the instrumentation. A disadvantage of glass tubes is the risk ofcontamination or exposure to pathogens in the event of breakage. Mostnon-breakable tubes are not suitable for autoclaving and for use inoptical detection systems since the container material interferes withthe detection system. Accordingly, there is a continuing need in theindustry for improved sample tubes.

SUMMARY OF THE INVENTION

The present invention is directed to a sample container suitable for usein an optical detection system. More particularly, the invention isdirected to a sample container made from a cyclic olefin copolymer. Theinvention is further directed to a method of detecting organisms usingthe sample container.

Accordingly, a primary object of the invention is to provide a samplecontainer for detecting the presence of microorganisms by fluorescenceanalysis.

Another object of the invention is to provide a non-breakable samplecontainer produced from a cyclic olefin copolymer that is opticallyclear to allow visual detection of a fluorescent compound by ultravioletlight and visual inspection of a microorganism growth medium.

A further object of the invention is to provide a sample container madefrom a cyclic olefin copolymer that is heat resistant to at least 250°C. without distortion or hazing of the container.

A still further object of the invention is to provide a sample containermade from a cyclic olefin copolymer that is able to withstand aninternal pressure of about 25 psi without rupturing or distortion.

Another object of the invention is to provide a sample container madefrom a cyclic olefin copolymer that is non-reactive with a liquidmicroorganism growth media and provides a shelf stable environment for amicroorganism growth media for extended periods of time.

These and other objects of the invention are basically attained byproviding a sample container assembly comprising a container having aside wall, a bottom wall, an open top end, and a liquid sample containedtherein, the container being formed from a cyclic olefin copolymerhaving a transparency sufficient to visually observe turbitity in saidsample; and a closure coupled to the open end of the container, whereinthe sample is substantially free of contamination upon prolonged storageof preferably about one year at 40° C.

The objects of the invention are further attained by providing amicrobial detecting container assembly comprising a transparentcontainer having a side wall, a bottom wall and an open top end, afluorescent sensor compound that exhibits a reduction in fluorescentintensity when exposed to oxygen, and a microorganism growth mediacontained within the container, the container being made by a moldingprocess from a cyclic olefin copolymer, wherein the container has atransparency sufficient to visually detect turbidity in themicroorganism growth media, and wherein the microorganism growth mediumis substantially free of contamination after storage for, preferably, atleast about 1 year at about 40° C.; and a closure coupled to the openend of the container.

The objects of the invention are also attained by providing a method ofdetecting the presence of microorganisms in a sample comprisingproviding a transparent container having a sidewall, a bottom wall, anopen top end, and a closure coupled to the open top end, the containercontaining a microorganism growth medium, wherein the container is madeby a molding process from a cyclic olefin copolymer, the containerhaving a transparency sufficient to visually detect turbitity in saidbacterial growth media and said bacterial growth medium beingsubstantially free of contamination after storage for, preferably, atleast about 1 year at 40° C., adding a sample to said transparentcontainer, irradiating said fluorescent sensor with a light sourcecapable of fluorescing said fluorescent sensor, detecting fluorescentlight intensity from said fluorescent sensor while irradiating saidfluorescent sensor, and comparing said detected fluorescent lightintensity with a control and determining the presence of microorganismsin the sample.

The objects advantages and other salient features of the invention willbecome apparent from the following detailed description of the inventionwhich taken in conjunction with the annexed drawings disclose variousembodiments of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

The following is a brief description of the drawings in which:

FIG. 1 is an exploded side elevational view of the container in apreferred embodiment of the invention; and

FIG. 2 is a side elevational view of the container of FIG. 1 showing afluorescent detection compound and a microorganism growth media solutioncontained therein.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to a container and to a method ofusing the container for detecting the presence of microorganisms. Moreparticularly, the invention is directed to a microbial detectingcontainer and to a method of detecting the presence of microorganisms ina sample within the container.

The container of the present invention can be a number of shapes andsizes depending on the type of sample being tested and theinstrumentation used for the sample analysis. In one preferredembodiment shown in FIGS. 1 and 2, the container 10 includes a tube 12having a generally cylindrical side wall 14 with a closed, roundedbottom 16. The tube 12 has an open top end 18 with external threads 20.A cap 22 having internal threads is provided for coupling with thethreads 20 of the tube 12 and forming a tight seal to prevent leakage ofthe contents. In one embodiment of the invention the tube has a capacityof about 12.0 ml with a length of about 10 cm and width of about 1.5 cm.In further embodiments, the shape and dimensions of the container canvary depending on the specific requirements of the analyzing procedure.

The container of the invention in a preferred embodiment is a sampletube for the detection of microorganisms by various methods, andparticularly by fluorescent measurements. For this purpose, the tube 12as shown in FIG. 2 includes a detecting compound 24, such as afluorescing compound, in the bottom wall 16. A microorganism nutrientgrowth media 26 is provided in the tube to support the growth of themicroorganism being detected.

The tube 14 is used for detecting the presence or absence of variousmicroorganisms, many of which are pathogenic. In one embodiment, thetube is used for detecting the presence of tuberculosis in a testsample. Therefore, the tube 14 is made from a non-breakable or fractureresistant polymeric material to minimize the risk of breakage and toreduce the risk of exposing the user to toxins or pathogens in thesample. It has been found that a tube made from a cyclic olefincopolymer has sufficient strength to resist breaking when dropped duringnormal use of the tube.

The container of the invention is required to be optically transparentor substantially transparent to allow visual inspection of the growthmedia contained therein. The container and growth media are required tobe shelf stable for extended periods of time, typically up to about oneyear. In preferred embodiments, the container is non-reactive with thegrowth media so that the growth media is free of suspended solids orother contaminants from the container after storage of about one year atabout 40° C. Turbidity of the growth medium is determined visuallythrough the transparent tube so that it is important to retain thetransparency throughout the storage life of the tube.

The sample tube is produced by molding the cyclic olefin copolymer usingknown molding processes depending on the desired shape and dimension ofthe sample tube. In one embodiment, the sample tube is made by aninjection molding process. Alternatively, the sample tube can be made byan injection blow molding process.

After the tube is molded, a fluorescent detecting compound is placed inthe bottom of the container and the container is filled with amicroorganism growth media. Typically the tube is cleaned and sterilizedbefore the fluorescent detecting compound and the growth media areplaced in the tube. After filling the tube with the microorganism growthmedia, the tube is closed by the threaded cap or other closure andautoclaved for sufficient time to sterilize the tube. Duringautoclaving, the tube is heated thereby causing an increase in theinternal pressure of the tube. In preferred embodiments, the sample tubewith the microorganism growth media is able to undergo autoclaving atleast at about 250° F. and is able to withstand an internal pressure ofat least 25 psi without rupturing, hazing, increasing brittleness of thetube, or contamination of the growth media by the tube material. In oneembodiment, the sample tube is sterilized by steam autoclaving. Afterautoclaving, the microorganism growth media stored in a cyclic olefinpolymer container is stable for at least one year and shows no turbitityor solid matter by visual inspection.

In preferred embodiments, the container is made from a cyclic olefincopolymer. These copolymers are thermoplastics suitable for injectionmolding or injection blow molding. The cyclic olefin copolymers havebeen found to have high transparency, good water vapor barrierproperties and heat resistance properties for use in producing thesample tubes for the fluorescence detection of microorganisms.

The cyclic olefin copolymers are block copolymers of a cyclic olefin andethylene having the formula

where R¹, R², R³, R⁴ and R⁵ and are the same or different and areselected from the group consisting of hydrogen and a C₁-C₈ alkylradical, and n and m are integers. In a preferred embodiment R⁴ and R⁵are hydrogen.

Particularly preferred cyclic olefin copolymers are produced from acyclic olefin and an acyclic olefin. Examples of preferred cyclic olefincopolymers are available under the trade name Topas (ThermoplasticOlefin Polymer of Amorphous Structure) from Hoechst Celanese of Summit,N.J. One preferred cyclic olefin copolymer is sold under the trade nameTopas-6015 having a light transmission of 92% determined by ASTM D-1003.Other suitable commercially available cyclic olefin copolymers areavailable under the trade names Topas-8007, Topas-5013 and Topas-6017.In one embodiment of the invention, the sample tube is made fromTopas-6015 by an injection blow molding process. The resin granules aredusted with an anti-blocking agent, such as Hoechstwax C sold by HoechstCelanese of Summit, N.J. The granules are dusted by tumbling theadditive in the form of a fine powder to cause the additive to adhere tothe resin granules. The resulting mixture is then fed into the screw ofan extruder of the molding machine in the usual manner. The additive andthe base resin are thoroughly mixed in passing through the extruder andreach the mold in the mixed state.

The cyclic olefin copolymers can be produced by various processes asknown in the art. For example, suitable cyclic olefin copolymers can beproduced by polymerizing about 0.1 to 100% by weight of a monomer havingthe formula

where R² and R⁹ are the same or different and are selected from thegroup consisting of hydrogen and a C₁-C₈ alkyl; up to 99.9% by weight ofa cyclic olefin having the formula

where n is an integer from 2 to 10; and up to 99.9% by weight of anacyclic olefin having the formula

wherein R¹⁰-R¹³ are the same or different and selected from the groupconsisting of hydrogen and C₁-C₈ alkyl. In the above formulas, the alkylcan be linear or branched.

The monomers are polymerized by a metallocene compound of group IVb toVb and an aluminoxane. Examples of processes for synthesizing the cyclicolefin copolymers are disclosed in U.S. Pat. Nos. 5,087,677, 5,331,057,5,324,801 and 5,422,409, which are hereby incorporated by reference intheir entirety.

Suitable cyclic olefin monomers for producing the copolymers include,for example, norborene, tetracyclododecene, bicyclo [2,2,1] hept-2-ene,1-methylbicyclo [2,2,1] hept-2-ene, and hexacyclo-4-heptadene. Thepreferred cyclic olefin can be copolymerized with an acyclic monomer,such as, ethylene, propylene, butylene or mixtures thereof. The cyclicolefin copolymers available under the trade name Topas are copolymers ofnorbene and ethylene.

The copolymers can be blended with various additives, such as antistaticagents, neutralizing agents, antioxidants, and organic peroxides asknown in the art to improve the processing properties and enhance thequalities of the finished product. Suitable antioxidants includephenolic antioxidants, phosphate antioxidants and organic phosphiteantioxidants. Typical neutralizing agents include stearic acid salts,oxides and hydroxides of alkaline earth metal.

The container of the invention is particularly suitable for a samplecontainer used in a method of measuring and detecting aerobicmicroorganisms by visually observing fluorescence of the sample. Toensure accurate results from the detection process, the sample tube mustretain its transparency after autoclaving and storage for prolongedperiods of time.

The fluorescent compound is a compound that is quenched at oxygenconcentrations normally found in a microorganism growth. media, which istypically a dissolved oxygen concentration in equilibrium with aheadspace oxygen concentration of about 7% to about 21%. Suitablefluorescent compounds include tris-2,2′bipyridyl ruthenium (II) saltssuch as the chloride salt Ru(DPP)₃Cl₂ and 9,10-diphenyl anthracene(DPA). The fluorescent compounds can be applied directly to the innersurfaces of the sample tube as a coating or combined within a suitablematrix that is applied to at least a portion of the inner surface of thesample tube. In a preferred embodiment the invention, the fluorescentcompound is dispersed throughout a silicone rubber that is permeable tooxygen. The silicone rubber is applied to the bottom of the sample tubeand cured. Preferably the fluorescent compound exhibits little or nofluorescence in the presence of oxygen.

A suitable microorganism growth medium is placed in the sample tube andthe tube is sealed by a suitable closure. The microorganism growthmedium can be selected depending on the microorganism being detected. Atypical growth medium can be a broth of, for example, Mueller Hinton II(BD Microbiology Systems), Brucella and Middlebrook 7H9 as known in theart. Typically, the sample tube contains about 4.0 ml to about 7.0 ml ofthe microorganism growth media. The microorganism does not fill thesample tube in preferred embodiments so a head space is present abovethe growth medium.

A sample suspected of containing a microorganism is added to the samplecontainer and the cap secured to the sample container to isolate theinterior from atmospheric oxygen. In alternative embodiments, the samplecontainer can be left open to the atmosphere. The sample container isthen incubated for sufficient period of time at a suitable incubatingtemperature for the specific microorganism being detected. Thereafterthe sample container is irradiated from a fluorescent light source,typically ultraviolet light, and the fluorescence of the fluorescentcompound detected. The intensity of the fluorescence is compared with acontrol to determine the presence of a microorganism. An example of thismethod is disclosed in U.S. Pat. No. 5,567,568, which is herebyincorporated by reference.

The fluorescence of the compound in the sample tube can be detected andmeasured using an automated testing apparatus. For example, the samplescan be analyzed using the detecting system sold under the trademarkBACTEC MGIT 960 for mycobacteria testing by Becton DickinsonMicrobiology Systems, Sparks, Md. Alternatively, the fluorescentemissions can be measured and detected using a Perkin-Elmer LS-SBequipped with a microtiter reader. In still further embodiments, theultraviolet light source can be a hand held unit and the fluorescenceobserved visually. The wavelength of the ultraviolet light can beselected depending on the particular fluorescent compound.

The process of the invention is particularly suitable for detecting thepresence of a variety of microorganisms and particularly mycobacteria.The sample container made from the cyclic olefin copolymer maintains itstransparency throughout the storage period and is sufficientlytransparent to provide accurate fluorescence measurement data. Thecyclic olefin copolymers can be steam autoclaved without increasing thebrittleness of the sample tube or affecting the gas and water barrierproperties. The cyclic olefin copolymers have a slight yellow color withvery low haze that does not interfere with fluorescent measurements orvisual detecting of tubitity of the growth media.

While various embodiments of the invention have been selected to showthe invention, it will be understood by those skilled in the art thatvarious modifications and changes can be made without departing from thescope of the invention.

What is claimed is:
 1. A method of determining the presence ofmicroorganisms in a sample comprising: providing a transparent containerhaving a sidewall, a bottom wall, an open top end, and a closure coupledto said open top end, said container containing a microorganism growthmedium, wherein said container is made by a molding process from cyclicolefin copolymer, said container having a transparency sufficient tovisually detect turbidity in said microorganism growth medium and saidmicroorganism growth medium being substantially free of contaminationafter storage for at least about one year at about 40° C.; adding asample suspected of containing a microorganism to said transparentcontainer; irradiating said fluorescent sensor with a light sourcecapable of fluorescing said fluorescent sensor; detecting fluorescentlight intensity from said fluorescent sensor while irradiating saidfluorescent sensor; and comparing said detected fluorescent lightintensity with a control and determining the presence of microorganismsin said sample.
 2. The method of claim 1, wherein said container has awall thickness sufficient to withstand an internal pressure of at least25 psi.
 3. The method of claim 1, wherein said container is able towithstand autoclaving at a temperature of at least 250° F.
 4. The methodof claim 1, wherein said fluorescent sensor compound comprises at leastone compound selected from the group consisting oftris-4,7-diphenyl-1,10-phenanthroline ruthenium (II) salts,tris-2,2′-bipyridyl ruthenium (II) salts, 9,10-diphenyl anthracene, andmixtures thereof.
 5. The method of claim 1, wherein said fluorescentsensor compound is tris-4,7-diphenyl-1,10-phenanthroline ruthenium (II)chloride.
 6. The method of claim 1, wherein said fluorescent sensorcompound is tris-2,2′-bipyridyl ruthenium (II) chloride hexahydrate. 7.The method of claim 1, wherein said cyclic olefin copolymer is acopolymer of a cyclic olefin and ethylene.
 8. The method of claim 1,wherein said cyclic olefin copolymer has the formula

where n and m are integers, R¹-R⁵ are the same or different and areselected from the group consisting of a hydrogen action and a C₁-C₈alkyl radical.